Brillouin scattering is the phenomena of inelastic light scattering induced by acoustic phonon of a material. In order to separate the small (typically in the order of GHz) Brillouin frequency shift from elastically scattered light, high-resolution spectrometer such as a multi-pass scanning Fabry-Perot interferometer is usually used in conventional Brillouin spectroscopy (Lindsay S M, Burgess S and Shepherd I W, “Correction of Brillouin linewidths measured by multipass Fabry-Perot spectroscopy,” Appl. Opt. 16(5), 1404-1407 (1977)). Since the dynamics of acoustic phonon is directly linked to the viscoelastic properties of a material, mechanical information can be acquired by measuring the Brillouin frequency shift of the scattered light (Dil J G, “Brillouin scattering in condensed matter,” Rep. Prog. Phys. 45, 286-334 (1982)). However, this method is fairly slow due to the point-by-point scan of the spectrum. In addition, the throughput efficiency is limited to the finesse of the etalon. This bottleneck was overcome by using a virtually imaged phased array (VIPA) etalon that can generate large angular dispersion (Xiao S, Weiner A M and Lin C, “A Dispersion Law for Virtually Imaged Phased-Array Spectral Dispersers Based on Paraxial Wave Theory,” IEEE J. Quantum Electronics 40(4), 420-426 (2004)) and enables measuring all of the spectral components simultaneously by a CCD camera with high throughput. Using this type of spectrometer, laser-scanning confocal Brillouin microscopy in biological tissue was performed at low illumination power and integration time, where each point in the sample is illuminated sequentially. The Brillouin spectrum was analyzed to create Brillouin-based elasticity maps (Scarcelli G and Yun S H, “Confocal Brillouin microscopy for three-dimensional mechanical imaging,” Nature Photonics 2, 39-43 (2007); Girard M J A, Dupps W J, Baskaran M, Scarcelli G, Yun S H, Quigley H A, Sigal I A and Strouthidis N G, “Translating Ocular Biomechanics into Clinical Practice: Current State and Future Prospects,” Curr. Eye Res. 40(1), 1-18 (2015)).
Brillouin spectroscopy allows non-invasive measurement of mechanical properties by measuring the frequency spectrum of acoustically-induced light scattering within a sample. Brillouin microscopy at sub-micron resolutions enabled the measurement of cell physico-chemical properties (Scarcelli G, Polacheck W J, Nia H T, Patel K, Grodzinsky A J, Kamm R D, Yun S H, “Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy”, Nat Methods. 12, 1132-1134 (2015)). In fact, cell mechanical properties critically regulate many cellular functions, e.g. proliferation, migration, gene expression as well as system-level behaviors, e.g. tissue morphogenesis, metastasis, and angiogenesis. As a result, “mechanical phenotyping”, i.e. the ability to classify cells based on their mechanical properties, has emerged as a powerful approach to characterize cell state and physiological/pathological conditions. For example, decreased cell stiffness has been shown to correlate with increased metastatic potential and thus it has been suggested as a novel label-free marker for tumor detection and staging (Swaminathan, V., et al. Mechanical Stiffness Grades Metastatic Potential in Patient Tumor Cells and in Cancer Cell Lines, Cancer Research 71, 5075-5080 (2011); Cross, S. E., Jin, Y. S., Rao, J. & Gimzewski, J. K. Nanomechanical analysis of cells from cancer patients. Nature Nanotechnology 2, 780-783 (2007)).
Current setups of Brillouin microscopy are usually based on epi-configuration, where the backward scattering light is collected by the same objective lens for illumination. One drawback of this configuration is that strong back-reflections will be coupled into the spectrometer as a background noise so that a spectrometer with high extinction ratio (usually two-stage VIPA) is required (Scarcelli, G. & Yun, S. H. Multistage VIPA etalons for high-extinction parallel Brillouin spectroscopy; Optics Express 19, 10913-10922 (2011)). In addition, since only one point at the focal plane of the objective lens can be measured each time, point-by-point scanning is needed for imaging purpose.
For cell mechanics, it is very important to achieve probing the subcellular mechanics in a non-contact, non-invasive manner, with high-resolution and high-density mapping and high-throughput. In fact, cells are highly heterogeneous, and thus subcellular information is crucial to fully understand the cells' biological activities. For example, the different behaviors of nucleus and cytoskeleton are highly relevant for several processes such as cell migration, proliferation, and cancerous cell migration during metastatic progression. Due to its size and high rigidity, nucleus imposes a major physical barrier for cells migration. The cytoskeleton, on the other hand, is a dynamic and adaptive 3D structure that fills the cytoplasm, and thus maintains the overall shape of the cell. During migration through 3D tissue, the movement of the nucleus must be coordinated with the cytoskeletal dynamics, and, as such, undergoes complex changes in position, stiffness, and shape, which in turn will affect the migration efficiency. When a normal cell transforms into a cancerous one, both changes in the cytoskeleton and nucleus will affect the ability for cells to attach and move. Therefore, the ability to characterize the stiffness of cytoskeleton and nucleus can result in potent mechanical biomarkers for the metastatic potential of cancer cells.
Accordingly, there is a need to integrate Brillouin microscopy with microfluidic technology to create a cytometry platform that classify cells without fluorescent labels by using intrinsic physical properties of the cell as contrast mechanism. Specifically, it is desired to obtain subcellular mechanical information from the spectral analysis of Brillouin light scattering that is related to the acoustic properties inside the cells. It is also desired to be able to classify living cells based on subcellular mechanical properties obtained by analyzing Brillouin scattering spectra generated from within the cells.
Furthermore, there is a need for a new Brillouin microscopy setup allowing for hundreds of points in a sample to be measured simultaneously using line-scanned parallel detection of scattering spectra. In other words, it is highly desirable to provide a Brillouin spectroscopy setup to obtain a high-resolution two-dimensional Brillouin image in a matter of seconds by scanning the sample only in one dimension.